U.S. patent number 4,446,126 [Application Number 06/357,504] was granted by the patent office on 1984-05-01 for antithrombin-heparin complex and method for its production.
This patent grant is currently assigned to Cutter Laboratories, Inc.. Invention is credited to Robert E. Jordan.
United States Patent |
4,446,126 |
Jordan |
May 1, 1984 |
Antithrombin-heparin complex and method for its production
Abstract
A complex of antithrombin and high activity heparin is provided
for use as a potent anticoagulant for humans. The complex is
prepared by reversibly immobilizing it on a lectin-containing,
water-insoluble gel matrix and then removing it from the
matrix.
Inventors: |
Jordan; Robert E. (Concord,
CA) |
Assignee: |
Cutter Laboratories, Inc.
(Berkeley, CA)
|
Family
ID: |
26887801 |
Appl.
No.: |
06/357,504 |
Filed: |
March 12, 1982 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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192170 |
Sep 30, 1980 |
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Current U.S.
Class: |
514/56; 514/14.7;
514/20.9; 514/822 |
Current CPC
Class: |
A61K
31/715 (20130101); C08B 37/0075 (20130101); Y10S
514/822 (20130101) |
Current International
Class: |
C08B
37/00 (20060101); C08B 37/10 (20060101); A61Y
031/725 (); A61Y 035/14 () |
Field of
Search: |
;424/101,183 |
Other References
Jordan et al, J. Biol. Chem. 254, 8, pp. 2902-2913, 4/25/79. .
Rosenberg et al., Proc. Nat. Acad. Sci., (1978), 75:7, 3065-3069.
.
Rosenberg et al., BBRL (1979), 86:4, 1319-1324..
|
Primary Examiner: Friedman; Stanley J.
Attorney, Agent or Firm: Leitereg; Theodore J. Aston; David
J. Johnson; Lester E.
Parent Case Text
This application is a continutation of application Ser. No.
192,170, filed Sept. 30, 1980, now abandoned.
Claims
I claim:
1. A method for preparing a composition having as its sole
effective anticoagulant agent an antithrombin-heparin complex,
comprising the steps of:
(a) reversibly binding antithrombin to a lectin-containing
water-insoluble matrix;
(b) contacting said matrix and said antithrombin bound thereto with
heparin, whereby an active portion of said heparin complexes with
said antithrombin;
(c) washing said matrix to remove portions of heparin not complexed
to said antithrombin;
(d) displacing said antithrombin-heparin complex from said matrix;
and
(e) thereby recovering said antithrombin-heparin complex
substantially free from uncomplexed antithrombin and uncomplexed
heparin.
2. The method of claim 1 wherein the lectin is Concanavalin A.
3. The method of claim 1 wherein the water-insoluble matrix is an
agarose gel.
4. The method of claim 1 wherein the unbound heparin is separated
from the matrix by treating the matrix with an aqueous salt
solution having an ionic strength sufficient to remove unbound
heparin but insufficient to remove high activity heparin from the
matrix.
5. The method of claim 1 wherein the matrix is contacted with an
aqueous carbohydrate solution, the carbohydrate being present in an
amount sufficient to separate the reversibly bound complex from the
matrix.
6. The method of claim 5 wherein the carbohydrate is a
monosaccharide.
7. The method of claim 6 wherein the carbohydrate is selected from
the group consisting of glucose, mannose, galactose, and
fructose.
8. The method of claim 5 wherein the carbohydrate is a
dissacharide.
9. The method of claim 8 wherein the carbohydrate is selected from
the group consisting of maltose, sucrose, and lactose.
10. A method of preparing a complex of antithrombin and high
activity heparin, which comprises
(a) contacting a lectin-containing, water-insoluble matrix with
antithrombin and heparin for a period of time and at a temperature
sufficient to allow the antithrombin to bind reversibly to the
lectin portion of the matrix and the high activity heparin
component of the heparin to complex with the antithrombin,
(b) washing the matrix with a solution having an ionic strength
sufficient to remove unbound heparin from the matrix but
insufficient to remove the antithrombin-high activity heparin
complex therefrom,
(c) contacting the matrix with a solution of a carbohydrate having
the ability to displace the complex from the matrix and
(d) recovering said complex substantially free of uncomplexed
antithrombin and uncomplexed heparin.
11. The method of claim 10 wherein 50-100 parts of matrix are
contacted in Step (a) with one part of antithrombin on a weight
basis.
12. The method of claim 10 wherein 1-100 parts of heparin per part
of antithrombin on a weight basis are contacted with the matrix in
Step (a).
13. The method of claim 10 wherein the matrix prior to contact with
antithrombin and heparin is equilibrated with a buffer solution
containing no greater than 0.25 M sodium chloride and having a pH
of about 6-8.5.
14. The method of claim 10 wherein the wash solution of Step (b)
has an ionic strength of about 0.1-0.4 and a pH of about 6-8.5.
15. The method of claim 10 wherein the temperature in Step (a) is
greater than 5.degree. C. but no greater than 37.degree. C.
16. The method of claim 10 wherein the time of contact in Step (a)
is about 0.1-2 hours.
17. The method of claim 10 wherein the matrix is contacted with a
solution in Step (c) containing 0.02-0.5 M carbohydrate and having
a pH of about 6-8.5.
18. The method of claim 10 wherein the carbohydrate is selected
from the group consisting of glucopyranosides, mannopyranosides,
fructofuranosides, monosaccharides, dissacharides, and sugar
alcohols.
19. The method of claim 10 which further includes the step of
reducing the carbohydrate concentration of the complex displaced
from the matrix in Step (c).
20. The method of claim 10 wherein the lectincontaining,
water-insoluble matrix in Step (a) is formed by covalently
attaching a lectin to a water-insoluble matrix.
21. A pharmaceutical preparation as an anticoagulant comprising a
complex of antithrombin and high activity heparin prepared by the
method of claim 10 wherein the activity of the high activity
heparin in the complex is greater than about 300 Units per
milligram and wherein the high activity heparin in the complex is
derived from heparin not previously fractioned by size.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention:
This invention relates to and has among its objects the provision
of a novel anticoagulant for human use. It is a particular object
of the invention to prepare an antithrombin-high activity heparin
complex in large quantities using a novel lectin-containing,
water-insoluble gel matrix to which antithrombin and heparin are
applied. Further objects of the invention will be evident from the
following description wherein parts and percentages are by weight
unless specified otherwise.
2. Description of the Prior Art:
Heparin is a glycosaminoglycan, having uronic acid, glucosamine,
and sulfate moieties, that functions as a blood anticoagulant by
binding to the inhibitor antithrombin and accelerating the rate at
which this inhibitor neutralizes serine proteases of the
coagulation mechanism.
The injection of unfractionated heparin alone (either intravenously
or subcutaneously) is routinely employed for the treatment of
thromboembolism or for the prevention of clot formation in at-risk
patients. Despite its widespread and growing use for these
purposes, problems concerning side effects and efficacy have been
pointed out. Some of the problems associated with current
anticoagulant therapy are the following:
(1) Patients often demonstrate widely different dose responses to
administered heparin. This requires a rather individualized
treatment procedure and constant monitoring of the resulting
clotting characteristics. Quite often, the desired dosage is
exceeded which necessitates the neutralization of the excess
heparin.
(2) Heparin, as it is used clinically, is approximately 30% active
as an anticoagulant (as defined by its ability to interact with
antithrombin which results in the rapid inhibition of thrombin and
other coagulation proteases). The majority of administered heparin
(70%) shows no particular affinity for antithrombin but can
interact with many other plasma proteins with consequences that may
be undesirable. The best example of this phenomenon is the
activation of lipoprotein lipase which results in the clearance of
circulating triglyceride.
(3) Heparin is a highly charged polyanion and is capable of many
non-specific electrostatic interactions with plasma proteins, blood
cells, and endothelial surfaces. Upon injection heparin becomes
distributed among these components. Although antithrombin binds in
a specific fashion and with high affinity to the active fraction of
the total heparin, is is unlikely that all of the anticoagulant
heparin binds to the plasma antithrombin. Thus, the actual
anticoagulant dosage of heparin received during heparin therapy is
a complex function of any number of equilibria which reduce the
amount of productive heparin-antithrombin complexes formed.
(4) Heparin has been implicated as a cause of thrombocytopenia due
to its interaction with platelets in patients undergoing prolonged
anticoagulant therapy.
(5) Circulating antithrombin levels have been shown to decrease as
a result of prolonged administration of heparin. Antithrombin
levels lowered in this way are reported to remain depressed for
several days following the end of treatment. This may be a
particularly undesirable effect in patients predisposed to
thrombosis.
(6) In patients with congenital antithrombin deficiency, the
administration of heparin may not be completely efficacious.
The administration of antithrombin as also been proposed to be a
means of controlling undesirable clot-formation in at-risk
patients. Those who might benefit most from this therapy would be
those congenitally deficient in antithrombin as well as individuals
undergoing certain types of surgery. In order for this type of
therapy to be effective, however, very large amounts of
antithrombin would be required. Also, treatment of congenital
antithrombin deficients with antithrombin concentrates would
require large amounts of this protein at frequent dosages since the
plasma half-life of antithrombin is about three days.
Fractionation of heparin into high and low activity components is
difficult because heparin species possessing active chain sequences
are virtually indistinguishable from those possessing inactive
chains. However, heparin has been separated into high activity and
low activity components by sucrose density gradient centrifugation
of heparin mixed with antithrombin-heparin cofactor (Lam et al,
Biochemical and Biophysical Research Communications, 1976, Vol. 69,
No. 2, pages 570-577). Heparin also has been fractionated by
affinity chromatography on immobilized antithrombin (Hook et al,
FEBS Letters, 1976, Vol. 66, pages 90-93). In this method
antithrombin is coupled covalently with a cyanogen
bromide-activated, water-insoluble matrix, such as, for example,
Sephadex.RTM., Sepharose.RTM., etc. Heparin is applied to the
immobilized antithrombin material, which adsorbs the high-activity
heparin species. After separation of the matrix containing the
adsorbed high activity component from the low activity heparin
component, the matrix is treated with a high salt medium to elute
the high activity heparin species therefrom.
An alternative method involves the separation of
heparin-antithrombin complexes from unbound heparin by gel
chromatography on Sephadex.RTM. G100. However, due to the size
heterogeneity inherent in commercial heparin preparations and the
resultingly broad chromatographic profile of the heparin itself,
the above method must employ heparin fractions of defined molecular
weight in order to permit the separation of the
heparin-antithrombin complex. This has been accomplished with a low
molecular weight heparin species having an average molecular weight
of 6000 daltons (Rosenberg et al, Proc. Nat. Acad. Sci., 1978, Vol.
75, No. 7, pages 3065-3069). In this case, a heparin-antithrombin
complex was separated from free heparin in an initial gel
chromatographic step and was subjected to a second chromatography
in the presence of high salt to disrupt the complex. The high
affinity heparin obtained in this sequence had a specific
anticoagulant activity of about 360 units/mg compared to the
starting pool of 96 units/mg. A low affinity heparin pool of 4
units/mg was also obtained by repetitive depletion of the starting
material.
A complex of antithrombin and high molecular weight, high affinity
heparin was also prepared by gel chromatography on Sephadex.RTM.
G100 (Rosenberg et al, B.B.R.C., 1979, Vol. 86, No. 4, pages
1319-1324). In this instance, the complex was separated from excess
antithrombin for the purpose of analytical characterization of the
ratios contained and required the use of a heparin species
previously fractionated both for size and activity.
One major problem confronting the industry in all of the
above-described methods is that the fractionation or preparation
has been accomplished only on a laboratory scale. In other words,
large scale manufacture or manufacture of pharmaceutically useful
amounts has not been realized either because of limitations
inherent in the method or because of the limited availability of
antithrombin. Compositions comprising a complex of antithrombin and
high activity heparin in a pharmaceutically useful amount,
essentially free of low activity heparin, the molecular weight of
the high activity heparin being representative of
non-fractionated-by-size heparin, have not been obtained in the
prior methods.
Fractionation of heparin into high and low activity components is
complicated further by the fact that antithrombin coupled to a
water-insoluble matrix cannot be recovered without substantial or
total destruction of the antithrombin. This results because the
antithrombin is covalently bound to the matrix by means of, for
example, a cyanogen bromide coupling process, and the conditions
necessary to cleave the coupling destroy the antithrombin.
Recently, a new method for the measurement of the binding of
ligands to solubilized membrane receptors, such as a receptor for
epidermal growth factor-urogastrone (EGF-URO) was described by Nexo
et al, J. Biol. Chem., 1979, Vol. 254, No. 18, pages 8740-8743. The
soluble receptor is first immobilized on lectin-agarose beads and
ligand binding is then determined on the bead-bound receptor. The
chromatographic and binding properties of solubilized receptor can
be studied due to the restoration of the ligand recognition
property of the receptor. After the solubilized receptor is
immobilized on lectin agarose, the binding of a ligand, such as
EGF-URO, to the immobilized receptor is rapid, reversible, peptide
specific, and of high affinity. The author notes that his method
deserves consideration for the study of any receptor that
recognizes a ligand free of carbohydrate.
SUMMARY OF THE INVENTION
I have found that a complex of antithrombin and high activity
heparin can be prepared by reversibly immobilizing the complex on a
lectin-containing, water-insoluble matrix. The antithrombin first
becomes reversibly bound to the matrix to which the high activity
component of heparin next becomes bound. The low activity or
unbound heparin may be separated selectively from the matrix which
then can be treated further to give the aforementioned complex. The
antithrombin and heparin can be applied to the matrix as a mixture,
or the antithrombin can be applied first followed by the heparin.
The so-prepared antithrombin-high activity heparin complex is
suitable for human use as an anticoagulant. It is to be noted that
use of any antithrombin-heparin complex as a therapeutic agent,
such as, for example, an anticoagulant, heretofore, has been
unknown in the art.
A primary advantage of the invention is that an
antithrombin-heparin complex can be prepared for therapeutic use in
large quantities by a quick and simple procedure. Thus, a valuable
therapeutic agent is now available in pharmaceutically useful
amounts; such an agent and its use are unknown in the prior art.
The antithrombin-heparin complex of the invention is a highly
potent therapeutic agent because the instant complex contains only
the high activity component of heparin. Prior to elution of the
antithrombin high activity heparin species from the matrix, the low
activity heparin species is removed.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the following description emphasis is directed to the
preparation of an antithrombin-HAH complex. Lectin-containing,
water-insoluble matrix is prepared from a water-insoluble polymeric
material and a lectin. As the water-insoluble polymeric material
one may use any material to which the lectin can be bound; thus,
one may use, by way of example and not limitation, certain
cross-linked dextrans, cross-linked agarose, etc. For instance, one
may employ Agarose or Sepharose.RTM. 4B, or the like. The lectin is
covalently bound to the matrix by means of cyanogen bromide or the
like, using the method described by Cuatrecasas, J. Biol. Chem.,
1970, Vol. 245, pages 3059-3065. It should be noted that any method
that covalently attaches a lectin to an insoluble matrix could be
used to prepare the matrix of this invention.
Lectins are carbohydrate-binding proteins of nonimmune origin that
agglutinate cells and/or precipitate complex carbohydrates and are
isolated usually from seeds of plants. The preferred lectin for
preparing the matrix for fractionating heparin is Concanavalin A.
However, other D-mannose (D-glucose)-binding lectins may be used
such as, for example, those described by Goldstein et al, in
Advances in Carbohydrate Chemistry and Biochemistry, 1978, Vol. 35,
pages 334-335.
The lectin-containing, water-insoluble matrix is mixed with
antithrombin which becomes reversibly bound to the matrix,
particularly to the lectin on the matrix. In a preliminary step,
which is optional although preferred, the lectin-containing,
water-insoluble matrix is equilibrated in an appropriate buffer
generally characterized as the same buffer solution as that used
hereinafter in which a mixture of antithrombin and heparin is
applied to the matrix. This buffer contains sodium chloride at a
level no greater than 0.25 M, preferably at physiological
concentration (0.15 M), and has a pH in the range of 6 to 8.5. The
equilibration is carried out for a period of about 0.1-2 hours at a
temperature of about 5-30.degree. C.
After the lectin-containing, water-insoluble matrix is
equilibrated, it is mixed with antithrombin, either pure or a
mixture with other proteins that will not bind to the lectin,
together with an excess of heparin, in an amount such that the
resultant system will contain high activity heparin (HAH). Usually,
about 50-1000 parts of matrix are mixed with one part of
antithrombin. By an excess of heparin is meant that about 1-100
parts, preferably 4-20 parts, of unfractionated heparin are
employed per part of antithrombin.
The mixture of antithrombin, heparin, and the lectin-containing
water-insoluble matrix is held in contact for a period of time and
at a temperature sufficient to allow the antithrombin to bind to
the lectin portion of the matrix. During that time the HAH becomes
complexed to the antithrombin. Thus, the mixture of antithrombin,
heparin, and the matrix are held for a period of about 0.1-2 hours
at a temperature compatible with the system, usually at a
temperature of about 5-30.degree. C. Generally, the antithrombin
and heparin are in solution in an appropriate buffer, preferably,
the buffering system employed in the above-described equilibration
of the lectin-containing, water-insoluble matrix; and the solution
is applied to a bed or column of the lectin-containing matrix.
Next, the matrix is washed to remove unbound heparin. The wash
solution should contain a physiologically acceptable salt having an
ionic strength sufficient to remove all unbound heparin from the
matrix but insufficient to remove the antithrombin-HAH complex,
preferably an ionic strength of about 0.1-0.4. The pH of the wash
solution should be about 6.0-8.5.
A suitable aqueous solution in accordance with this aspect of the
invention is, by way of example and not limitation, 0.1-0.4 M
sodium chloride (ionic strength=0.1-0.4) at pH 6.0-8.5. Ionic
strengths less than 0.1 should be avoided. Low ionic strength
promotes non-specific interactions between the lectin and heparin
itself. Low temperatures are to be avoided since such temperatures
also promote the above interactions. Thus, the preparation of the
complex should be conducted at a temperature greater than 5.degree.
C., preferably within the temperature range 20.degree.-30.degree.
C., and no greater than 37.degree. C. In general, the temperature
and ionic strength should be adjusted to achieve the appropriate
binding needed for selective complex formation where higher
temperatures require lower ionic strengths within the above
ranges.
In general, the matrix is washed until no unbound heparin appears
in the wash solution as determined by known methods.
The matrix, having been stripped of unbound heparin as described
above, may be treated to separate an antithrombin-HAH complex by
contacting the matrix with a solution of a carbohydrate having the
ability to displace the complex from the matrix. The concentration
of carbohydrate in the solution and the pH of the solution should
be sufficient to cause separation of the complex from the matrix.
Generally, about 0.02-0.5 M aqueous solution of carbohydrate at pH
6-8.5 is applied until the antithrombin is removed from the matrix.
As the carbohydrate one may use those carbohydrates disclosed by
Goldstein et al, supra, such as glucopyranosides, mannopyranosides,
and fructofuranosides. Mono- and disaccharides also may be employed
to separate the complex from the matrix and are preferred in this
particular step. Thus, one may use, by way of example and not
limitation, glucose, maltose, mannose, galactose, fructose,
lactose, sucrose, and the like. It is within the compass of the
invention to employ sugar alcohols such as mannitol, sorbitol, and
the like to isolate the aforementioned complex.
After separation of the complex, the eluted solution may be treated
to reduce its carbohydrate concentration by conventional means such
as by dialysis, diafiltration, etc., and then processed to put it
into condition for use. Generally, the eluate is concentrated also,
that is, treated to reduce its water content, by methods known in
the art for removing water from biologically active proteins
without reducing substantially their biological activity. The
concentrates may be sterilized by conventional means,
sterile-filtered, and treated to render them non-hepatitis
infective.
Antithrombin-HAH complex concentrates can be formulated into
pharmaceutical preparations. The term "pharmaceutical preparation"
is intended in a broad sense herein to include preparations used
for therapeutic purposes, for reagent purposes, for diagnostic
purposes, for tissue culture purposes, and so forth. The
pharmaceutical preparation intended for therapeutic use should
contain a therapeutic amount of the complex, i.e., that amount
necessary for preventative or curative health measures. If the
pharmaceutical preparation is to be employed as a reagent, then it
should contain reagent amounts of complex. Similarly, when used in
tissue culture or as a culture medium the pharmaceutical
preparation should contain an amount of complex sufficient to
obtain the desired growth. It is a characteristic of compositions
comprising the antithrombin-HAH complex prepared in accordance with
the present invention that they contain the complex in
pharmaceutically useful amounts. As mentioned earlier, an
antithrombin-heparin complex has been prepared only as an
intermediate in the laboratory scale production of heparin by gel
chromatography; consequently, compositions containing the complex
in pharmaceutically useful amounts have, heretofore, been unknown.
Furthermore, in the above heparin preparation using gel
chromatography, the heparin necessarily had to be fractionated by
size prior to use; and only fractionated-by weight heparin was
employed. The molecular weight of the high activity heparin in the
present complex is representative of non-fractionated-by-size
heparin, i.e., derived from heparin not previously fractionated by
size. It is also noteworthy that the instant complex is essentially
free of low activity heparin, and the activity of the high activity
heparin in the complex is greater than about 300 U/mg, usually
within the range of about 400-750 U/mg.
To prepare them for intravenous administration the compositions are
constituted usually in water containing physiologically compatible
substances such as sodium chloride, glycine, sugar and the like in
physiologically compatible concentrations and having a buffered pH
compatible with physiological conditions. Generally, guidelines for
intravenously administered compositions are established by
governmental regulations.
Since many of the problems and limitations of current heparin
therapy appear to result from inadequate levels of formed complexes
with circulating antithrombin, the administration of a preformed
HAH-antithrombin complex solves this problem. Thus, the levels of
HAH-antithrombin complex obtained would not be subject to the
conditions of the many equilibria which normally would prevent the
full anticoagulant expression of heparin administered alone.
Further, since no additional heparin would be given other than that
bound to the antithrombin, it is to be expected that side effects
resulting from the interaction of heparin with other serum
components would be greatly diminished. Also, in addition to the
increased potency and resulting lowered dosage of heparin required,
the dose response characteristics of the HAH-antithrombin
administration are expected to be much more predictable than with
current methods.
Two other advantages apply. First, circulating antithrombin levels
should not be decreased as a result of this anticoagulant therapy
since, presumably, only exogenously added antithrombin should be
complexed with HAH and thus subject to rapid clearing (heparin has
a circulating half-life of only 90 minutes). Second, administration
of antithrombin complexed with HAH should be beneficial to
congenital antithrombin deficients for whom complete restoration to
normal circulating levels may be economically difficult, and the
administration of heparin alone is ineffective.
It is also within the scope of the invention to initially
reversibly immobilize, in the absence of heparin, antithrombin on
the equilibrated lectin-containing, water-insoluble matrix from
above and use the resulting matrix to prepare the complex. Thus,
the matrix may be mixed with antithrombin, either pure or in a
mixture of other proteins that will not bind to the lectin on the
matrix, in an amount such that the resulting system will produce an
antithrombinHAH complex. About 50-1000 parts of matrix are mixed
usually with one part of antithrombin. The reaction conditions for
binding antithrombin to the matrix are the same as those described
above when heparin is present.
In situations where the antithrombin solution (in an appropriate
buffer system as described above) is not applied to a bed or column
of matrix, the matrix with bound antithrombin is treated to
separate it from the antithrombin solution. This may be
accomplished by techniques known in the art such as filtration,
decantation, and the like.
Next, the matrix is washed to remove residual antithrombin solution
and impurities not bound to the matrix. Preferably, the wash
solution is the same buffer solution described above in the
equilibration step.
It is characteristic of the aforedescribed system that the
antithrombin is bound reversibly thereto through the lectin on the
matrix. The antithrombin is bound sufficiently to immobilize it but
not great enough to cause the destruction of antithrombin upon its
removal from the matrix. Thus, the antithrombin is
non-destructively-removably bound to the lectin-containing matrix.
The exact nature of this reversible binding is not known. However,
hydrogen bonding and chargedipole interactions may be involved.
In the next step in this particular embodiment of the invention,
heparin is contacted with the antithrombincontaining,
lectin-containing, water-insoluble matrix onto which the high
activity heparin (HAH) is adsorbed. In general practice the
unfractionated heparin is in the form of a solution in a buffer
system containing sodium chloride in a concentration less than 0.4
M, usually about 0.1-0.4 M, and preferably at physiological
concentration, i.e., 0.15 M. The pH of the buffer solution should
be about 6.0-8.5, usually about 7.5. As the buffer solution one may
use, for example, a mixture of 0.01 M TRIS (hydroxymethyl)
aminomethane (TRIS) and 0.15 M sodium chloride. The amount of
heparin mixed with the matrix should be sufficient to maximize the
activity of HAH obtained in the complex, no less than about 1 part,
preferably 4-20 parts, of unfractionated heparin per part of
antithrombin on the matrix is applied to the matrix. The heparin
may be in pure form or it may be mixed with other proteins and the
like which do not bind to antithrombin in significant amounts.
Generally, contact between the heparin solution and the matrix is
achieved by forming a bed of freshly equilibrated matrix and
passing the heparin solution therethrough.
By the foregoing it is not meant to limit the invention to a
particular method of preparing the antithrombin-heparin complex of
the invention. I have discovered a novel therapeutic agent, namely,
an antithrombin-heparin complex and have developed also a novel
method for its production. Antithrombin-heparin complexes for
anticoagulant use obtained by other methods are within the scope of
my invention. Furthermore, previously fractionated heparin can also
be used in my method.
EXAMPLES
The invention is demonstrated further by the following illustrative
examples.
Assay Methods
Antithrombin III. The Lowry protein assay was used using human
serum albumin as the standard (Lowry et al, J. Biol. Chem., 1951,
Vol. 193, pages 165-275).
Heparin. Two assays were employed
(a) Carbazole Assay: A quantitative assay for heparin based on a
standard curve of uronic acid. A uronic acid content of 30% was
assumed for heparin (Hook et al, FEBS Letters, 1976, Vol. 66, pages
90-93). The assay was described by Bitter et al, in Anal.
Biochemistry, 1962, Vol. 4, pages 330-334.
(b) Azure A Method: A qualitative assay based on the method of
Jacques et al, J. Physiol. (London), 1949, Vol. 109, pages 41-48,
(see also Lam et al, BBRC, 1976, Vol. 69, pages 570-577).
Anticoagulant Activity of Heparin. The activity of all heparin
fractions was related to that of commercially obtained preparation
(Lipo-Hepin.RTM., Riker Laboratories, Inc.) whose U.S.P. unitage
was defined on the label. A standard curve was established with the
above heparin, and all heparin fractions of unknown activity were
determined by comparison to this curve by the following scheme:
(1) A 200 .mu.l sample containing antithrombin (approximately 30
.mu.g/ml) and 200 .mu.l of a heparin-containing solution were
combined and warmed to 37.degree..
(2) A 200 .mu.l sample of a solution containing thrombin
(Pentex.RTM. bovine thrombin, Miles Laboratories, Inc.) at a level
in excess of the antithrombin was added to the mixture of (1) above
and rapidly mixed.
(3) After exactly 30 seconds, 200 .mu.l of a solution of 1 mM
S-2238 (H-D-Phe-L-Pip-L-arg-p-nitroanilide, Kabi Diagnostica,
Sweden) and 0.5 mg Polybrene.RTM. (Aldrich Chemical Co., Inc.) was
added to the mixture which was again rapidly mixed.
(4) After exactly 60 seconds, 200 .mu.l of 50% acetic acid was
added to stop the esterolytic reaction.
(5) The U.V. absorbance of each sample was determined at 405
nanometers (nm).
EXAMPLE 1
Preparation of Antithrombin-Concanavalin A-Sepharose Support
A column (1.6.times.5 cm) was prepared from a 10 ml suspension of
Concanavalin A-Sepharose (Pharmacia Corp., Piscataway, N.J.). This
agarose gel contains 8 mg of the lectin protein covalently bound
per ml of swollen gel as stated by the manufacturer. The column was
equilibrated with a buffer containing 0.15 M NaCl and 0.01 M TRIS,
pH 7.5. (Optionally, this buffer solution can include agents to
prevent bacterial growth, e.g., sodium azide 0.02% and other salts
which stabilize Concanavalin A, namely, 0.1 mM calcium chloride and
0.1 mM manganese chloride, which are not otherwise essential for
the experiments to be described).
A solution containing antithrombin (0.83 mg/ml) in the above buffer
was applied to the column. Protein which was not bound to the
column was monitored by ultraviolet (UV) spectioscopy at 280 nm. An
unbound protein fraction was found to contain 1.13 mg which
represents approximately 9% of the applied material (12.5 mg).
Thus, approximately 11.4 mg of antithrombin was bound by the
Concanavalin A-Sepharose, which is approximately 1.4 mg
antithrombin per mg of Concanavalin A lectin.
EXAMPLE 2
Preparation of Antithrombin-HAH Complex
Heparin (85 mg) from Sigma Chemical Co., (St. Louis, Missouri) was
dissolved in 10 ml of an aqueous solution containing 0.15 M NaCl
and 0.01 M TRIS, pH 7.5, and the solution was applied to a column
prepared as in Example 1. The column was then eluted with an
aqueous buffer containing 0.01 M TRIS, pH 7.5, and 0.15 M NaCl
until no further heparin was detected in the eluate. The presence
of heparin in column eluates was monitored by a modification of the
Azure A Method in which the presence of the mucopolysaccharide
results in an increase in the visible absorption of the dye at 500
nm.
Next, the column was eluted with a neutral sugar dissolved in the
above buffer. In this case, a solution of 0.2 M
1-0-methyl-.alpha.-D-glucopyranoside was employed. The elution of
antithrombin-HAH from the column was detected by UV absorption at
280 nm and the Azure A Method described above.
The heparin contained in the above complex was shown to have high
activity, exhibiting a specific anticoagulant activity of 500
units/mg. Quantitation of the relative amounts of material
contained in the complex indicate that there is a molar excess of
antithrombin to heparin that is approximately two-fold. This value
is expected to vary between 1 to about 2.5 given the heterogeneous
size distribution of heparin and the ability of the larger species
to interact with more than one antithrombin molecule.
* * * * *